Antisense modulation of RhoB expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of RhoB. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding RhoB. Methods of using these compounds for modulation of RhoB expression and for treatment of diseases associated with expression of RhoB are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of RhoB. In particular, this invention relates toantisense compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding human RhoB. Sucholigonucleotides have been shown to modulate the expression of RhoB.

BACKGROUND OF THE INVENTION

In order to maintain their shape and integrity, it is critical that alltypes of cells contain a structural scaffold. This structure is known asthe cytoskeleton and is composed of a framework of interlockingproteins. The major protein component of the cytoskeleton is actin, andthe assembly of actin monomers into the cytoskeleton is highlyregulated.

Many cellular processes are mediated by the cytoskeleton. For example,changes occurring during cell cycle progression such as those associatedwith surface adhesion signals and the division of the cell into twodaughter cells (mitosis) are dependent on the appropriate assembly anddisassembly of the cytoskeleton. Therefore, it is currently believedthat the survival of the cell depends on the controlled regulation ofthe cytoskeleton.

RhoB, a member of the Rho subfamily of small GTPases, is a protein thathas been shown to be involved in a diverse set of signaling pathwaysincluding the regulation of the dynamic organization of the cytoskeletonduring the cell cycle.

Within the cell cycle, RhoB is first detected at the G₁ /S phasetransition, reaching a maximal level during the S phase. In addition,RhoB has been localized to early endosomes and pre-lysosomalcompartments within the cell. Since cytoskeletal modeling occurs duringthe S phase of the cell cycle and since RhoB is localized to membranousfractions, it has been suggested that RhoB plays a role in vesiculartraffic or the translocation of factors necessary during the cell cycle(Zalcman et al., Oncogene, 1995, 10, 1935-1945). In support of thishypothesis are recent studies showing that, in Swiss 3T3 cells, RhoBrecruits a protein kinase (PRK1) to endosomes (Mellor et al., J. Biol.Chem., 1998, 273, 4811-4814).

The RhoB gene has been classified as an immediate-early gene, whichmeans that its transcription is rapidly activated upon exposure tocertain growth factors or mitogens. The factors shown to activate RhoBtranscription include epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), genotoxic stress from UV light, alkylatingxenobiotics and the retroviral oncogene v-fps. Each of these stimulitriggers DNA synthesis in cultures of high cell density (Engel et al.,J. Biol. Chem., 1998, 273, 9921-9926). The response of RhoB to thesefactors implies a role for RhoB in wound repair and tissue regenerationupon growth factor stimulation and tumorigenesis upon mitogenstimulation.

Finally, manifestations of altered RhoB regulation also appear indisease states, including the development of cancer. Cellulartransformation and acquisition of the metastatic phenotype are the twomain changes normal cells undergo during the progression to cancer.Expression of constitutively activated forms of RhoB have been shown tocause tumorigenic transformation of NIH 3T3 and Ratl rodent fibroblasts(Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107). RhoB has alsobeen shown to be overexpressed in human breast cancer tissues (Zalcmanet al., Oncogene, 1995, 10, 1935-1945).

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of RhoB. To date, strategies aimed at inhibitingRhoB function have involved the use of bacterial enzymes such as theClostridium botulinum C3 exoenzyme which ADP ribosylates the proteinrendering it inactive or agents (natural enzyme inhibitors) to inhibitthe posttranslational modification (isoprenylation) of RhoB (Narumiyaand Morii, Cell Signal, 1993, 5, 9-19). However, these targetingstrategies are not specific to RhoB, as many proteins undergo similarposttranslational modifications. Consequently, there remains a long feltneed for additional agents capable of effectively inhibiting RhoBfunction.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to a nucleic acid encoding RhoB,and which modulate the expression of RhoB. Pharmaceutical and othercompositions comprising the antisense compounds of the invention arealso provided. Further provided are methods of modulating the expressionof RhoB in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of RhoB by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding RhoB, ultimately modulating the amountof RhoB produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingRhoB. As used herein, the terms "target nucleic acid" and "nucleic acidencoding RhoB" encompass DNA encoding RhoB, RNA (including pre-mRNA andmRNA) transcribed from such DNA, and also cDNA derived from such RNA.The specific hybridization of an oligomeric compound with its targetnucleic acid interferes with the normal function of the nucleic acid.This modulation of function of a target nucleic acid by compounds whichspecifically hybridize to it is generally referred to as "antisense".The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include allvital functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the expression of RhoB. In the context of the presentinvention, "modulation" means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. In the context of thepresent invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense."Targeting" an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding RhoB. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5'-AUG (in transcribed mRNAmolecules; 5'-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the "AUG codon," the "startcodon" or the "AUG start codon". A minority of genes have a translationinitiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, theterms "translation initiation codon" and "start codon" can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, "start codon" and "translationinitiation codon" refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding RhoB, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or"stop codon") of a gene may have one of three sequences, i.e., 5'-UAA,5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAGand 5'-TGA, respectively). The terms "start codon region" and"translation initiation codon region" refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationinitiation codon. Similarly, the terms "stop codon region" and"translation termination codon region" refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationtermination codon.

The open reading frame (ORF) or "coding region," which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5' untranslatedregion (5'UTR), known in the art to refer to the portion of an mRNA inthe 5' direction from the translation initiation codon, and thusincluding nucleotides between the 5' cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3' untranslated region (3'UTR), known in the art to refer to theportion of an mRNA in the 3' direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3' end of an mRNA or corresponding nucleotides onthe gene. The 5' cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5'-most residue of the mRNA via a 5'--5'triphosphate linkage. The 5' cap region of an mRNA is considered toinclude the 5' cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5' cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as "introns," which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as "exons" and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, "hybridization" means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. "Complementary," as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, "specifically hybridizable" and "complementary" are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans. In the context of this invention, the term"oligonucleotide" refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes oligonucleotides composed of naturally-occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases.Particularly preferred are antisense oligonucleotides comprising fromabout 8 to about 30 nucleobases (i.e. from about 8 to about 30 linkednucleosides). As is known in the art, a nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3'to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3'-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3'-5' linkages, 2'-5' linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂--N(CH₃)--O--CH₂ -- known as a methylene (methylimino) or MMI backbone!,--CH₂ --O--N(CH₃)--CH₂ --, --CH₂ --N(CH₃)--N(CH₃)--CH₂ -- and--O--N(CH₃)--CH₂ --CH₂ -- wherein the native phosphodiester backbone isrepresented as --O--P--O--CH₂ --! of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O (CH₂)_(n) O!_(m) CH₃,O(CH₂)_(n) OCH₃, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) ONH₂, andO(CH₂)_(n) ON (CH₂)_(n) CH₃)!₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2'position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2'-methoxyethoxy (2'--O--CH₂ CH₂ OCH₃,also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2'-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ ON(CH₃)₂ group, also known as 2'-DMAOE, as described in U.S.patent application Ser. No. 09/016,520, filed on Jan. 30, 1998, which iscommonly owned with the instant application and the contents of whichare herein incorporated by reference.

Other preferred modifications include 2'-methoxy (2'--O--CH₃),2'-aminopropoxy (2'--OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3' position of the sugar on the 3'terminal nucleotide or in 2'-5' linked oligonucleotides and the 5'position of 5' terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053l 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference and allowed U.S. patent application Ser. No. 08/468,037,filed on Jun. 5, 1995, U.S. Pat. No. X,XXX,XXX, which is commonly ownedwith the instant application and is also herein incorporated byreference.

Oligonucleotides may also include nucleobase (often referred to in theart simply as "base") modifications or substitutions. As used herein,"unmodified" or "natural" nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference, and U.S. Pat. No. 5,750,692, which iscommonly owned with the instant application and also herein incorporatedby reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. "Chimeric"antisense compounds or "chimeras," in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference, and allowed U.S.patent application Ser. No. 08/465,880, filed on Jun. 6, 1995, U.S. Pat.No. X,XXX,XXX, which is commonly owned with the instant application andalso herein incorporated by reference.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE(S-acetyl-2-thioethyl) phosphate! derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term "pharmaceutically acceptable salts" refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., "Pharmaceutical Salts," J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a "pharmaceutical addition salt"includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of RhoB is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingRhoB, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding RhoB can be detected by meansknown in the art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of RhoB in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2'-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).One or more penetration enhancers from one or more of these broadcategories may be included. Penetration enhancers are described inpending U.S. patent application Ser. No. 08/886,829, filed on Jul. 1,1997, and pending U.S. patent application Ser. No. 08/961,469, filed onOct. 31, 1997, both of which are commonly owned with the instantapplication and both of which are herein incorporated by reference.

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651-654). Examples of some presentlypreferred fatty acids are sodium caprate and sodium laurate, used singlyor in combination at concentrations of 0.5 to 5%.

Preferred penetration enhancers are disclosed in pending U.S. patentapplication Ser. No. 08/886,829, filed on Jul. 1, 1997, which iscommonly owned with the instant application and which is hereinincorporated by reference.

The physiological roles of bile include the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages934-935). Various natural bile salts, and their synthetic derivatives,act as penetration enhancers. Thus, the term "bile salt" includes any ofthe naturally occurring components of bile as well as any of theirsynthetic derivatives. Preferred bile salts are described in pendingU.S. patent application Ser. No. 08/886,829, filed on Jul. 1, 1997,which is commonly owned with the instant application and which is hereinincorporated by reference. A presently preferred bile salt ischenodeoxycholic acid (CDCA) (Sigma Chemical Company, St. Louis, Mo.),generally used at concentrations of 0.5 to 2%.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacids to make complex formulations. Preferred combinations include CDCAcombined with sodium caprate or sodium laurate (generally 0.5 to 5%).

Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 92-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; Buur et al., J.Control Rel., 1990, 14, 43-51). Chelating agents have the addedadvantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and perfluorochemical emulsions, such as FC-43 (Takahashi etal., J. Pharm. Pharmacol., 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, 8:2, 92-191); andnon-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

As used herein, "carrier compound" refers to a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioated oligonucleotide in hepatic tissue is reducedwhen it is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

In contrast to a carrier compound, a "pharmaceutically acceptablecarrier" (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of theinvention.

Regardless of the method by which the antisense compounds of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of thecompounds and/or to target the compounds to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layer(s) made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698-708).

Liposome preparation is described in pending U.S. patent applicationSer. No. 08/961,469, filed on Oct. 31, 1997, which is commonly ownedwith the instant application and which is herein incorporated byreference.

Certain embodiments of the invention provide for liposomes and othercompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Examples of antisenseoligonucleotides include, but are not limited to, those directed to thefollowing targets as disclosed in the indicated U.S. Patents, or pendingU.S. applications, which are commonly owned with the instant applicationand are hereby incorporated by reference, or the indicated published PCTapplications: raf (WO 96/39415, WO 95/32987 and U.S. Pat. No. 5,563,255,issued Oct. 8, 1996, and U.S. Pat. No. 5,656,612, issued Aug. 12, 1997),the p120 nucleolar antigen (WO 93/17125 and U.S. Pat. No. 5,656,743,issued Aug. 12, 1997), protein kinase C (WO 95/02069, WO 95/03833 and WO93/19203), multidrug resistance-associated protein (WO 95/10938 and U.S.Pat. No. 5,510,239, issued Mar. 23, 1996), subunits of transcriptionfactor AP-1 (pending application U.S. Ser. No. 08/837,201, filed Apr.14, 1997), Jun kinases (pending application U.S. Ser. No. 08/910,629,filed Aug. 13, 1997), MDR-1 (multidrug resistance glycoprotein; pendingapplication U.S. Ser. No. 08/731,199, filed Sep. 30, 1997), HIV (U.S.Pat. No. 5,166,195, issued Nov. 24, 1992 and 5,591,600, issued Jan. 7,1997), herpesvirus (U.S. Pat. No. 5,248,670, issued Sep. 28, 1993 andU.S. Pat. No. 5,514,577, issued May 7, 1996), cytomegalovirus (U.S. Pat.No. 5,442,049, issued Aug. 15, 1995 and U.S. Pat. No. 5,591,720, issuedJan. 7, 1997), papillomavirus (U.S. Pat. No. 5,457,189, issued Oct. 10,1995), intercellular adhesion molecule-1 (ICAM-1) (U.S. Pat. No.5,514,788, issued May 7, 1996), 5-lipoxygenase (U.S. Pat. No. 5,530,114,issued Jun. 25, 1996) and influenza virus (U.S. Pat. No. 5,580,767,issued Dec. 3, 1996). Two or more combined compounds may be usedtogether or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2'-alkoxy amidites

2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2'-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2'-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods Sanghvi, et.al., Nucleic Acids Research, 1993, 21, 3197-3203! using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2'-Fluoro Amidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthesized as described previouslyKawasaki, et. al., J. Med. Chem., 1993, 36, 831-841! and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2'-alpha-fluoro atom is introduced by a S_(N) 2-displacement of a2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and5'-DMT-3'-phosphoramidite intermediates.

2'-Fluorodeoxyguanosine

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5'-DMT- and5'-DMT-3'-phosphoramidites.

2'-Fluorouridine

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by themodification of a literature procedure in which2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5'-DMT and 5'-DMT-3'phosphoramidites.

2'-Fluorodeoxycytidine

2'-deoxy-2'-fluorocytidine was synthesized via amination of2'-deoxy-2'-fluorouridine, followed by selective protection to giveN4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used toobtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-O-(2-Methoxyethyl) modified amidites

2'-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2'-Anhydro 1-(beta-D-arabinofuranosyl)-5-methyluridine!

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂ Cl₂ /Acetone/MeOH (20:5:3)containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL)and adsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃ CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/Pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10° C., and the resulting mixture stirred foran additional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

A solution of3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃ NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2'-(Aminooxyethyl) nucleoside amidites and 2'-(dimethylaminooxyethyl)nucleoside amidites

Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per themethods of U.S. patent applications Ser. No. 10/037,143, filed Feb. 14,1998, and Ser. No. 09/016,520, filed Jan. 30, 1998, each of which iscommonly owned with the instant application and is herein incorporatedby reference.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hr), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the "gap" segment oflinked nucleosides is positioned between 5' and 3' "wing" segments oflinked nucleosides and a second "open end" type wherein the "gap"segment is located at either the 3' or the 5' terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas "gapmers" or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as "hemimers" or "wingmers".

2'-O-Me!-- 2'-deoxy!-- 2'-O-Me! Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and2'-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.oligonucleotides are synthesized using the automated synthesizer and2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2'-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 Ammonia/Ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2' positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to 1/2 volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

2'-O-(2-Methoxyethyl)!-- 2'-deoxy!-- 2'-O-(Methoxyethyl)! ChimericPhosphorothioate Oligonucleotides

2'-O-(2-methoxyethyl)!-- 2'-deoxy!-- -2'-O-(methoxyethyl)! chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2'-O-methyl chimeric oligonucleotide, with thesubstitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methylamidites.

2'-O-(2-Methoxyethyl)Phosphodiester!-- 2'-deoxy Phosphorothioate!--2'-O-(2-Methoxyethyl) Phosphodiester! Chimeric Oligonucleotides

2'-O-(2-methoxyethyl phosphodiester!-- 2'-deoxy phosphorothioate!--2'-O-(methoxyethyl) phosphodiester! chimeric oligonucleotides areprepared as per the above procedure for the 2'-O-methyl chimericoligonucleotide with the substitution of 2'-O-(methoxyethyl) amiditesfor the 2'-O-methyl amidites, oxidization with iodine to generate thephosphodiester internucleotide linkages within the wing portions of thechimeric structures and sulfurization utilizing 3,H-1,2benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate thephosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹ P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis--96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄ OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis--96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing four cell types are provided for illustrative purposes, butother cell types can be routinely used.

T-24 cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with antisense compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10 Analysis of Oligonucleotide Inhibition of RhoB Expression

Antisense modulation of RhoB expression can be assayed in a variety ofways known in the art. For example, RhoB mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions. Other methods of PCR are also known in the art.

RhoB protein levels can be quantitated in a variety of ways well knownin the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to RhoB can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total mRNA was isolated using an RNEASY ₉₆ ™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pippeting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

Example 13 Real-time Quantitative PCR Analysis of RhoB mRNA Levels

Quantitation of RhoB mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5' end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3' quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5'-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISM™ 7700 Sequence Detection System.In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). RhoB probes and primers were designedto hybridize to the human RhoB sequence, using published sequenceinformation (GenBank accession number X06820, incorporated herein as SEQID NO:1).

For RhoB the PCR primers were: forward primer: TGATCGTGTTCAGTAAGGACGAGTT(SEQ ID NO: 2) reverse primer: CGCCAGCTCCACCTGCTT (SEQ ID NO: 3) and thePCR probe was: FAM-TCTTCGAGAACTATGTGGCCGACATTGAG-TAMRA (SEQ ID NO: 4)where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

For GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQID NO: 5) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)and the PCRprobe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO: 7) whereJOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye.

Example 14 Northern Blot Analysis of RhoB mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST "B" Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions with a RhoB specific probe prepared by PCR usingthe forward primer TGATCGTGTTCAGTAAGGACGAGTT (SEQ ID NO: 2) and thereverse primer CGCCAGCTCCACCTGCTT (SEQ ID NO: 3). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA(Clontech, Palo Alto, Calif.). Hybridized membranes were visualized andquantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDHlevels in untreated controls.

Example 15 Antisense Inhibition of RhoB Expression--PhosphorothioateOligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human RhoB RNA, usingpublished sequences (GenBank accession number X06820, incorporatedherein as SEQ ID NO: 1). The oligonucleotides are shown in Table 1.Target sites are indicated by nucleotide numbers, as given in thesequence source reference (Genbank accession no. X06820), to which theoligonucleotide binds. All compounds in Table 1 areoligodeoxynucleotides with phosphorothioate backbones (internucleosidelinkages) throughout. The compounds were analyzed for effect on RhoBmRNA levels by quantitative real-time PCR as described in other examplesherein. Data are averages from three experiments. If present, "N.D."indicates "no data".

                  TABLE 1    ______________________________________    Inhibition of RhoB mRNA levels by phosphorothioate    oligodeoxynucleotides                                              SEQ                  TARGET               % In-  ID    ISIS #          REGION  SITE    SEQUENCE     hibition                                              NO.    ______________________________________    25384 Coding   14     ccaccaccagcttcttgc                                       0       8    25385 Coding   24     ccgtcgcccaccaccacc                                       0       9    25386 Coding   43     gcacgtcttgccacacgc                                       0      10    25387 Coding   61     actgaacacgatcagcag                                       0      11    25388 Coding   63     ttactgaacacgatcagc                                       0      12    25389 Coding   65     ccttactgaacacgatca                                       0      13    25390 Coding   67     gtccttactgaacacgat                                       5      14    25391 Coding   70     ctcgtccttactgaacac                                       1      15    25392 Coding   72     aactcgtccttactgaac                                       30     16    25393 Coding  110     catagttctcgaagacgg                                       0      17    25394 Coding  117     tcggccacatagttctcg                                       13     18    25395 Coding  132     ccgtccacctcaatgtcg                                       0      19    25396 Coding  234     aagcacatgagaatgacg                                       0      20    25397 Coding  255     gagtccgggctgtccacc                                       0      21    25398 Coding  267     atgttctccagcgagtcc                                       0      22    25399 Coding  270     gggatgttctccagcgag                                       33     23    25400 Coding  364     gacatgctcgtcgctgcg                                       0      24    25401 Coding  366     cggacatgctcgtcgctg                                       0      25    25402 Coding  370     tgtgcggacatgctcgtc                                       0      26    25403 Coding  373     ctctgtgcggacatgctc                                       39     27    25404 Coding  377     ccagctctgtgcggacat                                       21     28    25405 Coding  381     cgggccagctctgtgcgg                                       38     29    25406 Coding  383     tgcgggccagctctgtgc                                       31     30    25407 Coding  395     gttcctgcttcatgcggg                                       27     31    25408 Coding  399     acgggttcctgcttcatg                                       0      32    25409 Coding  451     gtagtcgtaggcttggat                                       29     33    25410 Coding  455     cgaggtagtcgtaggctt                                       39     34    25411 Coding  471     gtcttggcagagcactcg                                       20     35    25412 Coding  492     acctcgcgcacgccttcc                                       0      36    25413 Coding  494     agacctcgcgcacgcctt                                       16     37    25414 Coding  497     cgaagacctcgcgcacgc                                       0      38    25415 Coding  499     ctcgaagacctcgcgcac                                       0      39    25416 Coding  504     gccgtctcgaagacctcg                                       0      40    25417 Coding  508     cgtggccgtctcgaagac                                       0      41    25418 Coding  544     gttctgggagccgtagcg                                       36     42    25419 Coding  547     gccgttctgggagccgta                                       0      43    25420 Coding  553     gatgcagccgttctggga                                       0      44    25421 Coding  556     gttgatgcagccgttctg                                       7      45    25422 Coding  561     cagcagttgatgcagccg                                       31     46    25423 Coding  570     agcaccttgcagcagttg                                       0      47    ______________________________________

As shown in Table 1, SEQ ID NOs 16, 23, 27, 29, 30, 31, 33, 34, 42 and46 demonstrated at least 25% inhibition of RhoB expression in this assayand are therefore preferred.

Example 16 Antisense Inhibition of RhoB Expression--Phosphorothioate2'-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series ofoligonucleotides targeted to human RhoB were synthesized. Theoligonucleotide sequences are shown in Table 2. Target sites areindicated by nucleotide numbers, as given in the sequence sourcereference (Genbank accession no. X06820), to which the oligonucleotidebinds.

All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 18nucleotides in length, composed of a central "gap" region consisting often 2'-deoxynucleotides, which is flanked on both sides (5' and 3'directions) by four-nucleotide "wings". The wings are composed of2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide.Cytidine residues in the 2'-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from three experiments. If present,"N.D." indicates "no data".

                  TABLE 2    ______________________________________    Inhibition of RhoB mRNA levels by chimeric    phosphorothioate oligonucleotides having 2'-MOE     wings and a deoxy gap                                              SEQ                  TARGET               % In-  ID    ISIS #          REGION  SITE    SEQUENCE     hibition                                              NO.    ______________________________________    25424 Coding   14     ccaccaccagcttcttgc                                       29      8    25425 Coding   24     ccgtcgcccaccaccacc                                       23      9    25426 Coding   43     gcacgtcttgccacacgc                                       46     10    25427 Coding   61     actgaacacgatcagcag                                       37     11    25428 Coding   63     ttactgaacacgatcagc                                       47     12    25429 Coding   65     ccttactgaacacgatca                                        7     13    25430 Coding   67     gtccttactgaacacgat                                       46     14    25431 Coding   70     ctcgtccttactgaacac                                       52     15    25432 Coding   72     aactcgtccttactgaac                                       35     16    25433 Coding  110     catagttctcgaagacgg                                       29     17    25434 Coding  117     tcggccacatagttctcg                                       65     18    25435 Coding  132     ccgtccacctcaatgtcg                                       40     19    25436 Coding  234     aagcacatgagaatgacg                                       44     20    25437 Coding  255     gagtccgggctgtccacc                                       36     21    25438 Coding  267     atgttctccagcgagtcc                                       28     22    25439 Coding  270     gggatgttctccagcgag                                       54     23    25440 Coding  364     gacatgctcgtcgctgcg                                       49     24    25441 Coding  366     cggacatgctcgtcgctg                                       46     25    25442 Coding  370     tgtgcggacatgctcgtc                                       65     26    25443 Coding  373     ctctgtgcggacatgctc                                       39     27    25444 Coding  377     ccagctctgtgcggacat                                       19     28    25445 Coding  381     cgggccagctctgtgcgg                                       21     29    25446 Coding  383     tgcgggccagctctgtgc                                        9     30    25447 Coding  395     gttcctgcttcatgcggg                                       16     31    25448 Coding  399     acgggttcctgcttcatg                                        7     32    25449 Coding  451     gtagtcgtaggcttggat                                       38     33    25450 Coding  455     cgaggtagtcgtaggctt                                        0     34    25451 Coding  471     gtcttggcagagcactcg                                       42     35    25452 Coding  492     acctcgcgcacgccttcc                                        9     36    25453 Coding  494     agacctcgcgcacgcctt                                        7     37    25454 Coding  497     cgaagacctcgcgcacgc                                       12     38    25455 Coding  499     ctcgaagacctcgcgcac                                       23     39    25456 Coding  504     gccgtctcgaagacctcg                                       34     40    25457 Coding  508     cgtggccgtctcgaagac                                       27     41    25458 Coding  544     gttctgggagccgtagcg                                       58     42    25459 Coding  547     gccgttctgggagccgta                                       63     43    25460 Coding  553     gatgcagccgttctggga                                       17     44    25461 Coding  556     gttgatgcagccgttctg                                       21     45    25462 Coding  561     cagcagttgatgcagccg                                       50     46    25463 Coding  570     agcaccttgcagcagttg                                       55     47    ______________________________________

As shown in Table 2, SEQ ID NOs 10, 11, 12, 14, 15, 16, 18, 19, 20, 21,23, 24, 25, 26, 27, 33, 35, 40, 42, 43, 46 and 47 demonstrated at least30% inhibition of RhoB express ion in this experiment and are thereforepreferred.

Example 17 Western Blot Analysis of RhoB Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 hr after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to RhoB is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

    __________________________________________________________________________    #             SEQUENCE LISTING    - <160> NUMBER OF SEQ ID NOS: 47    - <210> SEQ ID NO 1    <211> LENGTH: 591    <212> TYPE: DNA    <213> ORGANISM: Homo sapiens    <220> FEATURE:    <221> NAME/KEY: CDS    <222> LOCATION: (1)..(591)    - <400> SEQUENCE: 1    - atg gcg gcc atc cgc aag aag ctg gtg gtg gt - #g ggc gac ggc gcg tgt      48    Met Ala Ala Ile Arg Lys Lys Leu Val Val Va - #l Gly Asp Gly Ala Cys    #                 15    - ggc aag acg tgc ctg ctg atc gtg ttc agt aa - #g gac gag ttc ccc gag      96    Gly Lys Thr Cys Leu Leu Ile Val Phe Ser Ly - #s Asp Glu Phe Pro Glu    #             30    - gtg tac gtg ccc acc gtc ttc gag aac tat gt - #g gcc gac att gag gtg     144    Val Tyr Val Pro Thr Val Phe Glu Asn Tyr Va - #l Ala Asp Ile Glu Val    #         45    - gac ggc aag cag gtg gag ctg gcg ctg tgg ga - #c acg gcg ggc cag gag     192    Asp Gly Lys Gln Val Glu Leu Ala Leu Trp As - #p Thr Ala Gly Gln Glu    #     60    - gac tac gac cgc ctg cgg ccg ctc tcc tac cc - #g gac acc gac gtc att     240    Asp Tyr Asp Arg Leu Arg Pro Leu Ser Tyr Pr - #o Asp Thr Asp Val Ile    # 80    - ctc atg tgc ttc tcg gtg gac agc ccg gac tc - #g ctg gag aac atc ccc     288    Leu Met Cys Phe Ser Val Asp Ser Pro Asp Se - #r Leu Glu Asn Ile Pro    #                 95    - gag aag tgg gtc ccc gag gtg aag cac ttc tg - #t ccc aat gtg ccc atc     336    Glu Lys Trp Val Pro Glu Val Lys His Phe Cy - #s Pro Asn Val Pro Ile    #           110    - atc ctg gtg gcc aac aaa aaa gac ctg cgc ag - #c gac gag cat gtc cgc     384    Ile Leu Val Ala Asn Lys Lys Asp Leu Arg Se - #r Asp Glu His Val Arg    #       125    - aca gag ctg gcc cgc atg aag cag gaa ccc gt - #g cgc acg gat gac ggc     432    Thr Glu Leu Ala Arg Met Lys Gln Glu Pro Va - #l Arg Thr Asp Asp Gly    #   140    - cgc gcc atg gcc gtg cgc atc caa gcc tac ga - #c tac ctc gag tgc tct     480    Arg Ala Met Ala Val Arg Ile Gln Ala Tyr As - #p Tyr Leu Glu Cys Ser    145                 1 - #50                 1 - #55                 1 -    #60    - gcc aag acc aag gaa ggc gtg cgc gag gtc tt - #c gag acg gcc acg cgc     528    Ala Lys Thr Lys Glu Gly Val Arg Glu Val Ph - #e Glu Thr Ala Thr Arg    #               175    - gcc gcg ctg cag aag cgc tac ggc tcc cag aa - #c ggc tgc atc aac tgc     576    Ala Ala Leu Gln Lys Arg Tyr Gly Ser Gln As - #n Gly Cys Ile Asn Cys    #           190    #   591            ga    Cys Lys Val Leu            195    - <210> SEQ ID NO 2    <211> LENGTH: 25    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: PCR Primer    - <400> SEQUENCE: 2    #               25 ggac gagtt    - <210> SEQ ID NO 3    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: PCR Primer    - <400> SEQUENCE: 3    #  18              tt    - <210> SEQ ID NO 4    <211> LENGTH: 29    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: PCR Probe    - <400> SEQUENCE: 4    #            29    ggcc gacattgag    - <210> SEQ ID NO 5    <211> LENGTH: 19    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: PCR Primer    - <400> SEQUENCE: 5    # 19               gtc    - <210> SEQ ID NO 6    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: PCR Primer    - <400> SEQUENCE: 6    # 20               tttc    - <210> SEQ ID NO 7    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: PCR Probe    - <400> SEQUENCE: 7    # 20               agcc    - <210> SEQ ID NO 8    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 8    #  18              gc    - <210> SEQ ID NO 9    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 9    #  18              cc    - <210> SEQ ID NO 10    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 10    #  18              gc    - <210> SEQ ID NO 11    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 11    #  18              ag    - <210> SEQ ID NO 12    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 12    #  18              gc    - <210> SEQ ID NO 13    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 13    #  18              ca    - <210> SEQ ID NO 14    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 14    #  18              at    - <210> SEQ ID NO 15    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 15    #  18              ac    - <210> SEQ ID NO 16    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 16    #  18              ac    - <210> SEQ ID NO 17    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 17    #  18              gg    - <210> SEQ ID NO 18    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 18    #  18              cg    - <210> SEQ ID NO 19    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 19    #  18              cg    - <210> SEQ ID NO 20    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 20    #  18              cg    - <210> SEQ ID NO 21    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 21    #  18              cc    - <210> SEQ ID NO 22    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 22    #  18              cc    - <210> SEQ ID NO 23    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 23    #  18              ag    - <210> SEQ ID NO 24    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 24    #  18              cg    - <210> SEQ ID NO 25    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 25    #  18              tg    - <210> SEQ ID NO 26    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 26    #  18              tc    - <210> SEQ ID NO 27    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 27    #  18              tc    - <210> SEQ ID NO 28    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 28    #  18              at    - <210> SEQ ID NO 29    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 29    #  18              gg    - <210> SEQ ID NO 30    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 30    #  18              gc    - <210> SEQ ID NO 31    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 31    #  18              gg    - <210> SEQ ID NO 32    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 32    #  18              tg    - <210> SEQ ID NO 33    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 33    #  18              at    - <210> SEQ ID NO 34    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 34    #  18              tt    - <210> SEQ ID NO 35    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 35    #  18              cg    - <210> SEQ ID NO 36    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 36    #  18              cc    - <210> SEQ ID NO 37    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 37    #  18              tt    - <210> SEQ ID NO 38    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 38    #  18              gc    - <210> SEQ ID NO 39    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 39    #  18              ac    - <210> SEQ ID NO 40    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 40    #  18              cg    - <210> SEQ ID NO 41    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 41    #  18              ac    - <210> SEQ ID NO 42    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 42    #  18              cg    - <210> SEQ ID NO 43    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 43    #  18              ta    - <210> SEQ ID NO 44    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 44    #  18              ga    - <210> SEQ ID NO 45    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 45    #  18              tg    - <210> SEQ ID NO 46    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 46    #  18              cg    - <210> SEQ ID NO 47    <211> LENGTH: 18    <212> TYPE: DNA    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <223> OTHER INFORMATION: Antisense Oligonucleotide    - <400> SEQUENCE: 47    #  18              tg    __________________________________________________________________________

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to SEQ ID NO:1, wherein said antisense compound inhibitsthe expression of human RhoB.
 2. The antisense compound of claim 1 whichis an antisense oligonucleotide.
 3. An antisense compound up to 30nucleobases in length comprising at least an 8-nucleobase portion of SEQID NO: 10, 11, 12, 14, 15, 16, 18, 19, 20, 21, 23, 24, 25, 26, 27, 29,30, 31, 33, 34, 35, 40, 42, 43, 46 or 47 which inhibits the expressionof human RhoB.
 4. The antisense compound of claim 3 comprising SEQ IDNO: 16, 23, 27, 33, 42 or
 46. 5. The antisense compound of claim 2 whichcomprises at least one modified internucleoside linkage.
 6. Theantisense compound of claim 5 wherein the modified internucleosidelinkage is a phosphorothioate linkage.
 7. The antisense compound ofclaim 2 which comprises at least one modified sugar moiety.
 8. Theantisense compound of claim 7 wherein the modified sugar moiety is a2'-O-methoxyethyl sugar moiety.
 9. The antisense compound of claim 2which comprises at least one modified nucleobase.
 10. The antisensecompound of claim 9 wherein the modified nucleobase is a5-methylcytosine.
 11. The antisense compound of claim 1 which is achimeric oligonucleotide.
 12. A method of inhibiting the expression ofhuman RhoB in human cells or tissues in vitro comprising contacting saidcells or tissues with the antisense compound of claim 1 so thatexpression of human RhoB is inhibited.